The overall goal of this proposal is to investigate the molecular and functional roles of long non-coding RNAs (lncRNAs) in regulating lung cell differentiation and development. Recent studies revealed that thousands of transcripts identified in mammalian cells correspond to lncRNAs. Similarly to protein-coding RNAs, lncRNAs can be tissue specific, highly regulated in development and altered in disease. Function of lncRNA in epigenetic gene regulation or in posttranscriptional events has been recently recognized. A significant number of lncRNAs originate by divergent transcription at promoters of genes encoding transcription factors and developmental regulators. These divergently transcribed lncRNA/mRNA pairs display similar expression patterns. Our goal is to define the mechanisms of action of lncRNAs divergently transcribed with lung developmental genes, and to evaluate their function in lung cell differentiation and development. We have preliminary evidence that the mouse Gata6AS is up-regulated similarly to Gata6 during ESCs differentiation, is co-immunoprecipitated with histone modifying proteins, and its down-regulation reduces the expression of the Gata6 mRNA. In supporting studies, we showed that human NKX2-1AS1 is expressed like NKX2-1 in primary lung epithelial but not mesenchymal cells and is localized in nucleoli and cytoplasm of lung carcinoma cells. Down-regulation of NKX2-1AS1 does not affect NKX2-1 mRNA expression but rather results in reduced cell proliferation and down regulation of cell cycle genes. We hypothesize that coordinated actions of lncRNA/mRNA pairs transcribed from lung developmental gene loci regulate lung epithelial cell differentiation. We will test this hypothesis in mouse ESCs differentiating in culture into endoderm and lung/thyroid progenitors and in mouse embryos. In Aim 1, we will analyze the effect of these lncRNAs on timing and efficiency of lung cell fate specification and differentiation in knock-down and over-expression experiments in ESCs in culture, and identify by RNA-sequencing other lncRNAs expressed in embryonic mouse lung, but not in thyroid or liver primordia. In Aim 2 we will determine the molecular role of Gata6AS, Nkx2-1AS, and other lung lncRNAs, by identifying interacting proteins by pull-down experiments followed by mass spectroscopy analysis. In Aim 3, we will test lncRNAs interacting with chromatin remodeling proteins by evaluating whether changes in expression levels of the lncRNA regulate downstream genes in cis or in trans by measuring changes in mRNA expression of nearby or distant genes by microarrays, binding of the corresponding proteins and lncRNAs to chromatin and alterations in histone marks in specific loci. For lncRNAs that interact with proteins involved in translation we will test protein levels o genes sharing complementary regions with the lncRNA in the same or distant loci. We will test the role of these lncRNAs in knock-down mice expressing shRNAs targeting the lncRNAs and evaluating the effect on lung organogenesis. Collectively, these studies will test for the first tie the functional role of lncRNA/mRNA divergent pairs in lung specific gene expression, cell differentiation and development.